Generation, characterisation and application of SARS-CoV-2 protein antigens for COVID-19 rapid diagnostic purposes in the hospital and community

我们的目标是培养一系列稳定转染的细胞系，以帮助满足对经过质量验证的重组SARS-CoV-2结构蛋白的需求，这些重组蛋白是我们迫切需要的，我们将使用它们来帮助开发诊断和疫苗。通过这样做，我们的项目将补充和扩展新冠肺炎蛋白质门户网站上提供的蛋白质库。我们建立的平台也将为未来快速产生其他目标做好完美的准备。我们将在两个不同的哺乳动物表达系统CHO和HEK293细胞中生产这些蛋白，通过质谱仪对其进行表征，并对Spike糖蛋白进行糖基化分析。在生产用于比较的非糖基化形式的蛋白质时，我们将确定这种翻译后修饰对折叠、三聚体形式组装的重要性，以及蛋白质检测康复的新冠肺炎患者抗尖峰抗体的能力。重组的真实和特有的SARS-CoV-2蛋白将在与我们的工业合作伙伴莫洛奇公司共同开发的诊断分析中进行评估，并将在位于阿什福德的东肯特医院信托基金的ELISA法中进行评估，以评估康复的新冠肺炎患者的抗体反应。这项拟议的研究将涉及世卫组织“2019年新型冠状病毒全球研究和创新论坛的全球协调研究路线图”，该路线图规定需要支持开发“可靠的血清学测试以及监测诊断产品的反应和中期支持开发的分析”的试剂，以改善临床过程，为控制/护理点做出贡献，并开发疫苗。

We aim to generate a range of stably transfected cell lines to help meet the demand for quality-verified recombinant SARS-CoV-2 structural proteins, which are desperately needed, and which we will use, to help in the development of diagnostics and vaccines. In so-doing our project will complement and expand the repertoire of proteins available on the COVID-19 Protein Portal. The platforms we establish will also be perfectly positioned for the rapid generation of other targets in the future. We will produce these in two different mammalian expression systems, CHO and HEK293 cells, characterise these by mass spectrometry and undertake glycosylation analysis of the Spike glycoprotein. In producing non-glycosylated forms of the protein for comparison, we will define the importance of this post-translational modification for folding, assembly of the trimer form and the ability of the protein to detect anti-spike antibodies from recovered COVID-19 patients. The recombinant authentic and characterised forms of the SARS-CoV-2 proteins will be assessed in diagnostic assays in development with our industrial partner Mologic, and in ELISA assays in the East Kent Hospital Trust at Ashford to profile antibody responses across recovered COVID-19 patients. The proposed research will address the WHO 'coordinated global research roadmap 2019 Novel Coronavirus Global Research and Innovation forum' that stipulates the need for reagents that support the development of 'reliable serological testing as well as assays that monitor response and midterm support development of diagnostic products to improve clinical processes to contribute to control/point of care' and for development of vaccines.